SRP438237 PRJNA973633 GSE232715 Employing totipotent cells to reconstruct embryogenesis [scRNA-seq] Blastoids, a structure similar to blastocysts in morphological and molecular level, can be applied to regeneration research. Using totipotent cells to construct blastoids will extend the information of early development to an earlier stage, and explore clues of regeneration. Totipotent blastomere-like cells (TBLCs) are a novel type of stably cultured mouse totipotent cell line generated by inhibiting spliceosomes. Here, we constructed blastoids (TBL-blastoids) in a new three-dimensional culture system using TBLCs. Morphological and transcriptomic analysis revealed TBL-blastoids contained typical morphology and key cell lineages of blastocysts and had higher degree of consistency in developmental rate and morphology compared to other blastoids. Moreover, TBL-blastoids implanted into uterus, induced decidua and even developed to embryonic tissues, indicating their in vivo developmental potential. The expansion and structures of TBL-blastoids in the IVC system also showed their in vitro developmental potential. The efficiency of generating TBL-blastoids and implantation rate suggest the necessity of TE-like component formation. Meanwhile, TBLCs can differentiate into extraembryonic cell lines directly, which provides an alternative strategy for evaluating totipotency. Furthermore, we explored the impacts of senescence, a central role in regeneration, on TBLCs and found that cellular senescence impaired the totipotency of TBLCs and the efficiency of generating blastoids. Also, the in vivo and in vitro developmental potential of TBL-blastoids were declined. In conclusion, the induction of TBLCs into blastoids and extraembryonic cells is valuable for promoting regeneration, early embryonic development study and evaluating totipotency. Overall design: We constructed blastoids (TBL-blastoids) in a new three-dimensional culture system using TBLCs. Morphological and transcriptomic analysis revealed TBL-blastoids contained typical morphology and key cell lineages of blastocysts and had higher degree of consistency in developmental rate and morphology compared to other blastoids. Sample 1 is the TBLC cell line derived from miPSCs treated with spliceosomal inhibitor. TBLCs are the totipotent-like cells which resembles 2-cell embryonic cells and possesses totipotent features. Sample 2 and Sample 3 are synthetic embryos and constructed by TBLCs. TBL-blastoids have the typical morphology and key cell lineages of blastocysts and are blastocyt-like structures. Sample 2 and Sample 3 are the biological replicates to perform scRNA-seq. Sample 4 are also the TBL-blastoids and were derived from the TBLCs treated with 1 mM H2O2 for 1 h. GSE232715 pubmed 38402947 parent_bioproject PRJNA973624